1. Field of the Invention
This invention resides in the field of laboratory equipment for electrophoretic separations, and the particular equipment addressed herein is that designed for isoelectric focusing.
2. Background of the Invention
As is well known in the art, isoelectric focusing is the separation of proteins into a linear array according to their isoelectric points and is performed on a sample for purposes of detection, analysis, and in some cases quantification of the proteins in the sample. Isoelectric focusing is typically performed in gel strips that contain immobilized pH gradients (“IPG strips”), and can serve either as the entire separation process of the proteins, or as the first dimension of a two-dimensional separation. In two-dimensional separations, the second dimension is performed by placing the IPG strip, after the first dimension separation has been performed, along one edge of a two-dimensional (“slab”-shaped) separation medium and imposing an electric field on the two-dimensional medium in a direction transverse to the IPG strip. This causes the proteins in each focused zone in the IPG strip to migrate into, and spread out within, the slab, thereby extending the linear array in the IPG strip to a two-dimensional array. The proteins in the original sample thus have the benefit of being separated according to two parameters. Descriptions of isoelectric focusing and some of the equipment in which isoelectric focusing is performed can be found in Panattoni, “Assembly for Casting and Use of an Isoelectric Focusing Strip,” U.S. Pat. No. 6,655,649, issue date Dec. 2, 2003; Williams et al., “Apparatus for Electrophoresis,” U.S. Pat. No. 6,558,522, issue date May 6, 2003; Faupel et al., “Isoelectric Focusing Apparatus,” U.S. Pat. No. 5,082,548, issue date Jan. 21, 1992; and Witkorowicz et al., “Electrophoresis Apparatus and Method,” U.S. Pat. No. 6,214,191, issue date Apr. 10, 2001.
IPG strips are commercially available from suppliers to biochemical laboratory suppliers and are commonly supplied in dehydrated form to be rehydrated by the user prior to use in the separation procedure. Both rehydration and focusing are typically performed in trays that accommodate as many as 24 IPG strips for simultaneous separations of as many samples. In some procedures, rehydration is performed in a rehydration/equilibration tray and once rehydrated, the strips are transferred to a separate focusing tray. In other procedures, rehydration and focusing are performed in the same tray. When the same tray, or any tray with electrodes contacting the strips, is used, rehydration can be performed in the presence of an electric current (“active rehydration”). This is particularly useful when the sample is combined with the rehydration solution and contains high molecular weight proteins, since the current enhances the entry of the proteins into the IPG strip. In certain separations, the sample is loaded through a separate receptacle at one end of the IPG strip, i.e., by “cup loading,” while in others the sample is applied to the entire length of the strip.
The typical IPG strip obtained from a supplier is a thin, flat strip of gel material that has a backing on one side for dimensional stability and ease of handling and a protective sheet on the other side. The protective sheet is removed by the user prior to rehydration and discarded, while the backing remains in place throughout both the rehydration and focusing. The strip thus contains a “gel side” and a backing side, and with the gel in a horizontal position the gel side can be facing either up or down during the rehydration step, and either up or down during the focusing step, depending on the needs of the sample to be separated, the preference of the user, or both. During focusing and active rehydration, however, the electrodes must be in contact with the gel side. The trays presently available are designed for use with gels in either one orientation or the other, i.e., trays intended for contact of the electrodes with gels whose gel sides are facing up are distinct in their design and construction from trays intended for contact of the electrodes with gel sides facing down.